The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H₂O₂)-mediated cross-linking and degradation of the polymers by varying the exposure time to H₂O₂ contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H₂O₂ exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H₂O₂-mediated degradation. These results indicate that H₂O₂-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Engineering skeletal muscle tissue in vitro is important to study the mechanism of myogenesis, which is crucial for regenerating muscle cells. The physicochemical properties of the cellular microenvironment are known to govern various cell behaviours. Yet, most studies utilised synthetic materials to model the extracellular matrix that suffers from cytotoxicity to the cells. We have previously reported that the physicochemical property of hydrogels obtained from horseradish peroxidase (HRP)-catalysed cross-linking could be controlled by a simple adjustment to the exposure time to air containing H₂O₂. In this study, we evaluated the influence of physicochemical properties dynamics in the gelatin possessing phenol groups (Gelatin-Ph) hydrogel to regulate the myogenesis in vitro. We controlled the Young’s modulus of the Gelatin-Ph hydrogel by tuning the air containing 16 ppm H₂O₂ exposure time for 15–60 min. Additionally, prolonged exposure to air containing H₂O₂ also induced Gelatin-Ph degradation. Myoblasts showed higher adhesion and myotube formation on stiff hydrogel (3.53 kPa) fabricated through 30 min of exposure to air containing H₂O₂ compared to those on softer hydrogel (0.77–2.79 kPa) fabricated through 15, 45, and 60 min of the exposure. These results demonstrate that the myogenesis can be tuned by changes in the physicochemical properties of Gelatin-Ph hydrogel mediated by H₂O₂.

The development of hydrogels that allow vascular endothelial cells to form capillary-like networks is critical for advancing tissue engineering and drug discovery. In this study, we developed hydrogels composed of phenolated hyaluronic acid (HA-Ph) with an average molecular weight of 490–159 kDa via sonication in an aqueous solution. These hydrogels were synthesized by the horseradish peroxidase-catalyzed crosslinking of phenol moieties in the presence of hydrogen peroxide and phenolated gelatin. The sonication-degraded HA-Ph (198 kDa) significantly enhanced the migration ability of human umbilical vein endothelial cells (HUVECs) on cell culture plates when added to the medium compared to the original HA-Ph (490 kDa) and less-degraded HA-Ph (312–399 kDa). In addition, HUVECs cultured on these hydrogels formed networks that did not occur on hydrogels made from the original HA-Ph. CD44 expression and PI3K gene expression, both markers related to angiogenesis, were 3.5- and 1.8-fold higher, respectively, in cells cultured on sonication-degraded HA-Ph hydrogels than in those cultured on hydrogels comprising the original HA-Ph. These results highlight the potential of hydrogels containing sonication-degraded HA-Ph for tissue engineering and drug-screening applications involving human vascular endothelial cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA‐Ph) and phenolated gelatin (Gelatin‐Ph) are presented. Visible‐light irradiation in the presence of tris(2,2′‐bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA‐Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible‐light irradiation for 45 min (42 W cm⁻² at 450 nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60 min. The irradiation time‐dependent degradation of HA‐Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and β‐actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA‐Ph degradation. These findings highlight the potential of tailoring HA‐based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.